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Thesis PF.Indb Studies on biological functions of the transcription factors Sp3 and Sp4 Pieter Fokko van Loo © P.F. van Loo All rights reserved. No part of this book may be reproduced or transmitted, in any form or by any means, without written permission of the author ISBN: 90-5335-0500 Printed by Ridderprint offsetdrukkerij B.V., Ridderkerk Studies on biological functions of the transcription factors Sp3 and Sp4 Onderzoek naar biologische functies van de transcriptiefactoren Sp3 en Sp4 Proefschrift ter verkrijgen van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de Rector Magnifi cus Prof. dr. S.W.J. Lamberts en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 13 april 2005 om 15.45 uur door Pieter Fokko van Loo geboren te Hoogeveen. Promotiecommssie Promotor: Prof. dr. F.G. Grosveld Overige leden: Prof. dr. D.J.G.M. Duncker Prof. dr. J.A. Grootegoed Prof. dr. C.P. Verrijzer Copromotor: Dr. J.N.J. Philipsen Dit proefschrift is tot stand gekomen binnen de Vakgroep Celbiologie aan de Faculteit der Geneeskunde en Gezondheidswetenschappen van de Erasmus MC Medische Universiteit Rotterdam. De Vakgroep maakt deel uit van het Medisch Genetisch Centrum Zuid-West Nederland. Het onderzoek is fi nancieel ondersteund door de Nederlandse Organisatie voor Wetenschappelijk onderzoek NWO. Het verschijnen van dit proefschrift in boekvorm is mede mogelijk gemaakt door een fi nanciële bijdrage van J.E. Jurriaanse Stichting. 2 In het begin schiep God de hemel en de aarde. Genesis 1 vers 1 (nieuwe bijbel vertaling) Contents Abbreviations page 6 Scope of this thesis page 9 Chapter 1 Introduction page 11 Chapter 2 Impaired hematopoiesis in mice lacking the transcription factor Sp3 page 41 Chapter 3 Transcription factor Sp3 has a crucial role in cardiac development page 61 Chapter 4 Identifi cation of target genes of the transcription factor Sp3 in fetal page 79 murine heart by microarray experiments Chapter 5 Sexual behaviour and odour preference in Sp4 knockout mice page 99 Chapter 6 Discussion: concluding remarks and future outlook page 117 Samenvatting page 129 Curriculum vitae page 135 Nawoord page 137 5 Abbreviations A anterior aaRNA aminoallyl RNA Abca8b ATP-binding cassette, sub-family A 8b Abdh1 alpha/beta hydrolase domain containing 1 AER apical ectodermal ridge AGM aorta-gonads-mesonephros Amhc1 atrial myosin heavy chain 1 Ao aorta AP2rep activating protein 2 repressor Aprt adenine phosphoribosyl transferase Bhlh basic helix-loop-helix bp base pair(s) Bklf basic klf Btd-box Buttonhead-box Bteb basic transcription element binding protein cAMP cyclic adenosine mono phosphate cDNA complementary DNA CNS central nervous system Cnx connexin CLP common lymphoid progenitor CMP common myeloid progenitor Coup TFII chicken ovalbumin upstream promoter transcription factor II DC dendritic cell Defcr-rs12 defensin related cryptin, related sequence 12 DILV double inlet left ventricle DNA deoxyribonucleic acid dNTP deoxy-N-5’-triphosphate DORV double outlet right ventricle DTT 1,4-dithiothreitol Egf(r) epidermal growth factor (receptor) EMP erythrocyte-megakaryocyte progenitor Eklf erythroid klf EPDC epicardial derived cell ErbB see Egfr ER estrogen receptor ES cell embryonic stem cell ET1 endothelin1 Fgf fi broblast growth factor Fog1 friend of Gata1 Gklf gut klf GMP granulocyte-monocyte progenitor GTF general transcription factor H2A histone type 2A H2B histone type 2B H3 histone type 3 H4 histone type 4 HPC hematopoietic progenitor cell HSC hematopoietic stem cell Iklf intestine enriched klf Irx4 iroquois related homeobox 4 Kklf kidney klf klf Krüppel-like factor L left LA left atrium Lcn5 lipocalin 5 Lklf lung klf LV left ventricle 6 mBtd mouse Buttonhead mRNA messenger RNA Mef2c myocyte enhancer factor 2c Mlc2a myosin light chain 2 atrial Mlc2v myosin light chain 2 ventricular Nhlh2 nescient helix-loop-helix 2 NK cell natural killer cell O-GlcNAc O-linked monosaccharide β-N-acetylglucosamine P posterior PA pulmonary artery Pax5 paired box gene 5 Pdgfβ platelet derived growth factor β chain PEST proline, glutamine, serine and threonine enriched sequences Pka protein kinase A Pkcγ protein kinase C γ R right RA right atrium RNA ribonucleic acid RNApI RNA polymerase I RNApII RNA polymerase II RNApIII RNA polymerase III rRNA ribosomal RNA RV right ventricle Rxrα retinoic x receptor α SDS sodium dodecyl sulfate Slc6a20 solute carrier family 6 member 20 Sp specifi city protein Spock1 sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 Srebp sterol regulatory element binding protein SUMO small ubiquitin-like modifi er SV40 Simian virus 40 T Brachyury mutation TAF TBP-associated factor TAFII110 TATA-box binding protein-associated factor II 110 TBP TATA-box binding protein Tbx5 T-box transcription factor 5 TCRα T-cell antigen receptor α TFIIX transcription factor IIX Tgfβ transforming growth factor β Th1 T helper 1 Th2 T helper 2 Tieg tgf β inducible early growth response protein Tpk1 thiamin pyrophosphokinase 1 tRNA transfer RNA TSA histone deacetylase inhibitor trichostatin A Ttf1 tfhyroid transcription factor 1 Ug/Ccsp uteroglobin/clara cell secretory protein U units Uklf ubiquitous klf UTP uridine-5’-triphosphate Vegf vascular endothelial growth factor Vmhc1 ventricle specifi c myosin heavy chain 1 VNO vomeronasal organ VSD ventricular septal defect XKLF x Krüppel-like factor Ypel1 yippee-like 1 Zf9 zinc-fi nger protein 9 Znf714 zinc-fi nger protein 741 7 Scope of this thesis In this thesis I describe the work that I have executed to study functions of the transcription factors Sp3 and Sp4. Previous experiments on these factors have shown that Sp3 is absolutely essential for normal embryogenesis and that Sp4 is required for post-natal functioning and survival, including sexual behaviour. However, their exact biological roles, and how they fulfi l them, remain a mystery. The experiments described in this thesis were designed to come closer to the answers of these questions. Chapter 1 To place the transcription factors Sp3 and Sp4 in the biological framework of transcription, the fi rst chapter introduces the Sp/XKLF family and in particular the Sp subfamily, and describes the role of transcription factors in the regulation of eukaryotic gene transcription in general, as well as in hematopoiesis and cardiac development. Chapter 2 Sp3 has been functionally associated with the hematopoietic system. This chapter describes our study that was undertaken to charaterize the role that Sp3 fulfi ls in the hematopoietic system. This study was performed by an analysis of fetal and adult hematopoiesis in Sp3-defi cient mice. Chapter 3 The defect that causes the perinatal death of the Sp3-/- mice remains unknown. As preliminary observations gave a hint that cardiac dysfunction might bring about their perinatal death, we undertook morphological and molecular analysis of Sp3-/- fetal hearts. Chapter 4 To understand the role of Sp3 in embryogenesis and survival, target genes of Sp3 have to be identifi ed. To identify such genes, we studied and compared the gene expression profi les of fetal Sp3+/+ and Sp3-/- hearts by microarray experiments. Chapter 5 To understand the role of Sp4 in sexual maturation, the impaired sexual behaviour of Sp4-defi cient males and females was studied in detail by behavioural- and molecular studies. Chapter 6 Many pieces remain in the puzzle of the biological functions of Sp3 and Sp4. This chapter is a concluding remark on the Sp3 and Sp4 data presented here, and gives an outlook on new experiments that may contribute to elucidate further aspects of the biological roles of Sp3 and Sp4. 9 Chapter 1 Introduction Transcription is regulated by transcription factors Eukaryotic genomes typically contain thousands of genes harbouring information for the proteins that regulate cellular processes. To utilize the encrypted genetic information, genes have to be transcribed into messenger RNA (mRNA), which thereupon is translated into proteins. Transcription of genes into mRNA is highly regulated in time and space to ensure that produced proteins can fulfi l their biological role in the development or maintenance of cells and body at the right place and at the right time. In eukaryotes, the transcription of genes into mRNA is performed by the RNA polymerase II (RNApII) enzyme. The two other eukaryotic polymerases, RNApI and RNApIII, transcribe ribosomal RNA (rRNA) and transfer RNA (tRNA), respectively.1 The activity of RNApII is regulated by a large protein complex of general transcription factors (GTF), e.g. transcription factor IIB (TFIIB) and TFIID.2 A representative example of regulation of RNApII during initiation and prolongation of transcription is the interaction of RNApII to promoters containing a TATA-box, an AT-rich sequence located 25-30 bp upstream the transcriptional initiation site. The RNApII is recruited to the TATA-box containing promoter by an association between RNApII and the multi-subunit complex TFIID. A subunit of the TFIID complex, the TATA-box binding protein (TBP), facilitates fi nal binding of the entire protein complex, which includes RNApII, to the TATA-box. Other well known TFIID subunits are the TBP-associated factors (TAFs).3,4 However, the basal transcriptional machinery alone is not suffi cient to initiate or to direct transcription effi ciently. Discovery of sequence-specifi c DNA-binding proteins or transcription factors revealed that RNApII transcription is directed by cis control elements present in proximal promoter and distal enhancer regions in the genome. Via binding to the cis control elements and via subsequent recruiting of cofactors (corepressors or coactivators) these factors direct the activity of basal transcription machinery and thereby contribute to correct transcription of a gene (review see references 5,6). Specifi city of expression of each individual gene is accomplished by complex arrays of various transcription factor target sequences in the cis control elements of the gene.7 DNA in the cell is packaged into chromatin. One of the tasks of transcription factors is to recruit chromatin modifi ers to the DNA to modify the condensation grade of chromatin.
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